Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation.

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.

Store-Operated Ca2+ Channels: Mechanism, Function, Pharmacology, and Therapeutic Targets.

Calcium (Ca2+) release-activated Ca2+ (CRAC) channels are a major route for Ca2+ entry in eukaryotic cells. These channels are store operated, opening when the endoplasmic reticulum (ER) is depleted of Ca2+, and are composed of the ER Ca2+ sensor protein STIM and the pore-forming plasma membrane subunit Orai. Recent years have heralded major strides in our understanding of the structure, gating, and function of the channels. Loss-of-function and gain-of-function mutants combined with RNAi knockdown strategies have revealed important roles for the channel in numerous human diseases, making the channel a clinically relevant target. Drugs targeting the channels generally lack specificity or exhibit poor efficacy in animal models. However, the landscape is changing, and CRAC channel blockers are now entering clinical trials. Here, we describe the key molecular and biological features of CRAC channels, consider various diseases associated with aberrant channel activity, and discuss targeting of the channels from a therapeutic perspective.

Control of NFAT Isoform Activation and NFAT-Dependent Gene Expression through Two Coincident and Spatially Segregated Intracellular Ca2+ Signals.

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.

The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+ entry.

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca2+ nanodomain, which forms when Ca2+ channels open. Ca2+ nanodomains arising from store-operated Orai1 Ca2+ channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca2+ entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca2+ channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca2+ channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.

House dust mite allergens, store-operated Ca2+ channels and asthma.

The house dust mite is the principal source of aero-allergen worldwide. Exposure to mite-derived allergens is associated with the development of asthma in susceptible individuals, and the majority of asthmatics are allergic to the mite. Mite-derived allergens are functionally diverse and activate multiple cell types within the lung that result in chronic inflammation. Allergens activate store-operated Ca2+ release-activated Ca2+ (CRAC) channels, which are widely expressed in multiple cell types within the lung that are associated with the pathogenesis of asthma. Opening of CRAC channels stimulates Ca2+ -dependent transcription factors, including nuclear factor of activated T cells and nuclear factor-κB, which drive expression of a plethora of pro-inflammatory cytokines and chemokines that help to sustain chronic inflammation. Here, I describe drivers of asthma, properties of mite-derived allergens, how the allergens are recognized by cells, the signalling pathways used by the receptors and how these are transduced into functional effects, with a focus on CRAC channels. In vivo experiments that demonstrate the effectiveness of targeting CRAC channels as a potential new therapy for treating mite-induced asthma are also discussed, in tandem with other possible approaches.

Distinct spatial Ca2+ signatures selectively activate different NFAT transcription factor isoforms.

Protein isoforms are widely expressed in biological systems. How isoforms that co-exist within the same sub-cellular domain are differentially activated remains unclear. Here, we compare the regulatory mechanism of two closely related transcription factor isoforms, NFAT1 and NFAT4, that migrate from the cytoplasm to the nucleus following the increase in intracellular Ca(2+) that accompanies the opening of store-operated Orai1/CRAC channels. We demonstrate that NFAT1 has a private line of communication with Orai1, activating in response to Ca(2+) microdomains near the open channels. By contrast, NFAT4 stimulation requires both local Ca(2+) entry and a nuclear Ca(2+) rise. We mapped differences in nuclear location to amino acids within the SP-3 motif of the NFAT regulatory domain. The different Ca(2+) dependencies enable agonists to recruit different isoform combinations as stimulus strength increases. Our study uncovers a mechanism whereby co-existing cytoplasmic transcription factor isoforms are differentially activated by distinct sub-cellular Ca(2+) signals.

Single-nucleotide polymorphisms in Orai1 associated with atopic dermatitis inhibit protein turnover, decrease calcium entry and disrupt calcium-dependent gene expression.

Loss-of function mutations in Orai1 Ca2+ channels lead to a form of severe combined immunodeficiency, auto-immunity, muscle hypotonia and defects in dental enamel production and sweat gland function. Two single-nucleotide polymorphisms (SNPs) in Orai1 have been found and localize to the second extracellular loop. These polymorphisms associate with atopic dermatitis but how they affect Ca2+ signalling and cell function is unknown. Here, we find that Orai1-SNPs turnover considerably more slowly than wild type Orai1 and are more abundantly expressed in the plasma membrane. We show a central role for flotillin in the endocytotic recycling of Orai1 channels and that endocytosed wild type Orai1 is trafficked to Rab 7-positive late endosomes for lysosomal degradation. Orai1-SNPs escape the degradation pathway and instead enter Rab 11-positive recycling endosomes, where they are returned to the surface membrane through Arf6-dependent exocytosis. We find that Orai1-SNPs escape late endosomes through endosomal pH regulation of interaction between the channel and flotillin. We identify a pH-sensitive electrostatic interaction between positively charged arginine in extracellular loop 2 (K210) and a negatively charged aspartate (D112) in extracellular loop 1 that helps determine Orai1 turnover. The increase in membrane Orai1-SNP leads to a mis-match in Orai1-STIM stoichiometry, resulting in inhibition of Ca2+ entry and Ca2+-dependent gene expression. Our results identify new strategies for targeting atopic dermatitis.

The whole-cell Ca2+ release-activated Ca2+ current, ICRAC , is regulated by the mitochondrial Ca2+ uniporter channel and is independent of extracellular and cytosolic Na.

KEY POINTS: Ca2+ entry through Ca2+  release-activated Ca2+  channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca2+ buffering, mitochondrial Ca2+ uptake regulates CRAC channel activity. Knockdown of the mitochondrial Ca2+ uniporter channel prevented the development of ICRAC in weak buffer but not when strong buffer was used instead. Removal of either extracellular or intra-pipette Na+ had no effect on the selectivity, kinetics, amplitude, rectification or reversal potential of whole-cell CRAC current. Knockdown of the mitochondrial Na+ -Ca2+ exchanger did not prevent the development of ICRAC in strong or weak Ca2+ buffer. Whole cell CRAC current is Ca2+ -selective. Mitochondrial Ca2+ channels, and not Na+ -dependent transport, regulate CRAC channels under physiological conditions. ABSTRACT: Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels plays a central role in activation of a range of cellular responses over broad spatial and temporal bandwidths. Mitochondria, through their ability to take up cytosolic Ca2+ , are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca2+ buffering. The mitochondrial Ca2+ transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca2+ uniporter (MCU) channel or the Na+ -Ca2+ -Li+ exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca2+ transporters in supporting the CRAC current (ICRAC ) under a range of conditions in RBL mast cells. Knockdown of the MCU channel impaired the activation of ICRAC under physiological conditions of weak intracellular Ca2+ buffering. In strong Ca2+ buffer, knockdown of the MCU channel did not inhibit ICRAC development demonstrating that mitochondria regulate CRAC channels under physiological conditions by buffering of cytosolic Ca2+ via the MCU channel. Surprisingly, manipulations that altered extracellular Na+ , cytosolic Na+ or both failed to inhibit the development of ICRAC in either strong or weak intracellular Ca2+ buffer. Knockdown of NCLX also did not affect ICRAC . Prolonged removal of external Na+ also had no significant effect on store-operated Ca2+ entry, on cytosolic Ca2+ oscillations generated by receptor stimulation or on CRAC channel-driven gene expression. In the RBL mast cell, Ca2+ flux through the MCU but not NCLX is indispensable for activation of ICRAC .

Cysteinyl leukotriene type I receptor desensitization sustains Ca2+-dependent gene expression.

Receptor desensitization is a universal mechanism to turn off a biological response; in this process, the ability of a physiological trigger to activate a cell is lost despite the continued presence of the stimulus. Receptor desensitization of G-protein-coupled receptors involves uncoupling of the receptor from its G-protein or second-messenger pathway followed by receptor internalization. G-protein-coupled cysteinyl leukotriene type I (CysLT1) receptors regulate immune-cell function and CysLT1 receptors are an established therapeutic target for allergies, including asthma. Desensitization of CysLT1 receptors arises predominantly from protein-kinase-C-dependent phosphorylation of three serine residues in the receptor carboxy terminus. Physiological concentrations of the receptor agonist leukotriene C(4) (LTC(4)) evoke repetitive cytoplasmic Ca(2+) oscillations, reflecting regenerative Ca(2+) release from stores, which is sustained by Ca(2+) entry through store-operated calcium-release-activated calcium (CRAC) channels. CRAC channels are tightly linked to expression of the transcription factor c-fos, a regulator of numerous genes important to cell growth and development. Here we show that abolishing leukotriene receptor desensitization suppresses agonist-driven gene expression in a rat cell line. Mechanistically, stimulation of non-desensitizing receptors evoked prolonged inositol-trisphosphate-mediated Ca(2+) release, which led to accelerated Ca(2+)-dependent slow inactivation of CRAC channels and a subsequent loss of excitation-transcription coupling. Hence, rather than serving to turn off a biological response, reversible desensitization of a Ca(2+) mobilizing receptor acts as an 'on' switch, sustaining long-term signalling in the immune system.

Spatial Ca2+ profiling: decrypting the universal cytosolic Ca2+ oscillation.

Stimulation of cell-surface receptors that couple to phospholipase C to generate the second messenger inositol trisphosphate often evokes repetitive oscillations in cytosolic Ca2+ . Signalling information is encoded in both the amplitude and frequency of the Ca2+ spikes. Recent studies have revealed that the spatial profile of the oscillation also imparts signalling power; Ca2+ microdomains near store-operated CRAC channels in the plasma membrane and inositol trisphosphate-gated channels in the endoplasmic reticulum both signal to distinct downstream targets. Spatial profiling therefore increases the transduction power of the universal oscillatory cytosolic Ca2+ signal.

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors.

Oscillations in cytoplasmic Ca(2+) concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca(2+) oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca(2+)-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca(2+) oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li(+), an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li(+)-treated cells, cytoplasmic Ca(2+) signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca(2+) oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca(2+) oscillations, and these could not be rescued by inositol or PI4P. In Li(+)-treated cells, recovery of the cytoplasmic Ca(2+) oscillations in the presence of inositol or PI4P was suppressed when Ca(2+) influx through store-operated Ca(2+) channels was inhibited. After rundown of the Ca(2+) signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca(2+) release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca(2+) entry is needed to sustain cytoplasmic Ca(2+) signaling following leukotriene receptor activation both by refilling the Ca(2+) stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.

Decoding cytosolic Ca2+ oscillations.

A rise in cytosolic Ca(2+) concentration is used as a universal signalling mechanism to control biological processes as diverse as exocytosis, contraction, cell growth and cell death. Ca(2+) signals are often presented to cells in the form of Ca(2+) oscillations, with signalling information encoded in both amplitude and frequency of the Ca(2+) spikes. Recent studies have revealed that the sub-cellular spatial profile of the Ca(2+) oscillation is also important in activating cellular responses, thereby suggesting a new mechanism for extracting information from the ubiquitous Ca(2+) oscillation.

Caveolin-1 alters the pattern of cytoplasmic Ca2+ oscillations and Ca2+-dependent gene expression by enhancing leukotriene receptor desensitization.

Cytoplasmic Ca(2+) oscillations constitute a widespread signaling mode and are often generated in response to stimulation of G protein-coupled receptors that activate phospholipase C. In mast cells, repetitive Ca(2+) oscillations can be evoked by modest activation of cysteinyl leukotriene type I receptors by the physiological trigger, leukotriene C4. The Ca(2+) oscillations arise from regenerative Ca(2+) release from inositol 1,4,5-trisphosphate-sensitive stores followed by Ca(2+) entry through store-operated Ca(2+) channels, and the latter selectively activate the Ca(2+)-dependent transcription factor NFAT. The cysteinyl leukotriene type I receptors desensitize through negative feedback by protein kinase C, which terminates the oscillatory Ca(2+) response. Here, we show that the scaffolding protein caveolin-1 has a profound effect on receptor-driven Ca(2+) signals and downstream gene expression. Overexpression of caveolin-1 increased receptor-phospholipase C coupling, resulting in initially larger Ca(2+) release transients of longer duration but which then ran down quickly. NFAT-activated gene expression, triggered in response to the Ca(2+) signal, was also reduced by caveolin-1. Mutagenesis studies revealed that these effects required a functional scaffolding domain within caveolin-1. Mechanistically, the increase in Ca(2+) release in the presence of caveolin-1 activated protein kinase C, which accelerated homologous desensitization of the leukotriene receptor and thereby terminated the oscillatory Ca(2+) response. Our results reveal that caveolin-1 is a bimodal regulator of receptor-dependent Ca(2+) signaling, which fine-tunes the spatial and temporal profile of the Ca(2+) rise and thereby its ability to activate the NFAT pathway.

Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression.

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.

Scrutinizing science to save lives: uncovering flaws in the data linking L-type calcium channels blockers to CRAC channels and heart failure.

Hypertension is estimated to affect almost 1 billion people globally and significantly increases risk of myocardial infarction, heart failure, stroke, retinopathy and kidney disease. One major front line therapy that has been used for over 50 years involves L-type Ca 2+ channel blockers (LCCBs). One class of LCCBs is the dihydropyridine family, with amlodipine being widely prescribed regardless of gender, race, ethnicity or age. In 2020, Johnson et al. 7 reported that all LCCBs significantly increased the risk of heart failure, and attributed this effect to non-canonical activation of store-operated Ca 2+ entry. A major approach on which they based many of their arguments was to measure cytosolic Ca 2+ using the fluorescent Ca 2+ indicator dye fura-2. We recently demonstrated that amlodipine is highly fluorescent within cells and overwhelms the fura-2 signal, precluding the use of the indicator dye with amlodipine 24 . Our meta-analyses and prospective real world study showed that dihydropyridines were not associated with an increase in heart failure, likely explained by the lack of consideration by Johnson et al. 7 of well-known confounding factors such as age, race, obesity, prior anti-hypertensive treatment or diabetes 24 . Trebak and colleagues have responded to our paper with a forthright and unwavering defence of their work 27 . In this paper, we carry out a forensic dissection of Johnson et al., 7 and conduct new experiments that address directly points raised by Trebak et al. 27 . We show that there are major flaws in the design and interpretation of their key experiments, that fura-2 cannot be used with amlodipine, that there are fundamental mathematical misunderstandings and mistakes throughout their study leading to critical calculations on heart failure that are demonstrably wrong, and several of their own results are inconsistent with their interpretation. We therefore believe the study by Johnson et al. 7 is flawed at many levels and we stand by our conclusions.

CRAC channels and Ca2+ signaling in mast cells.

Mast cells are integral members of the immune system. Upon activation by a rise in cytoplasmic Ca2+, they release a battery of paracrine signals, chemokines, and cytokines, which help sculpt the subsequent immune response. Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane is central for driving most of these responses. The molecular basis of the CRAC channel has been identified, with Orai1 forming the channel pore. Recent work has revealed that a range of mast cell responses are activated by spatially restricted Ca2+ signals just below the plasma membrane. These Ca2+ microdomains can activate cytosolic enzymes, leading to the generation of intracellular messengers as well as proinflammatory molecules like LTC4. In this review, we describe key features of CRAC channels in mast cells, how they generate local Ca2+ signals, and how the cell can decode these restricted signals to generate a raft of responses.

A Reappraisal of the Effects of L-type Ca2+ Channel Blockers on Store-Operated Ca2+ Entry and Heart Failure.

Dihydropyridines such as amlodipine are widely used as antihypertensive agents, being prescribed to ∼70 million Americans and >0.4 billion adults worldwide. Dihydropyridines block voltage-gated Ca2+ channels in resistance vessels, leading to vasodilation and a reduction in blood pressure. Various meta-analyses show that dihydropyridines are relatively safe and effective in reducing hypertension. The use of dihydropyridines has recently been called into question as these drugs appear to activate store-operated Ca2+ entry in fura-2-loaded nonexcitable cells, trigger vascular remodeling, and increase heart failure, leading to the questioning of their clinical use. Given that hypertension is the dominant "silent killer" across the globe affecting ∼1.13 billion people, removal of Ca2+ channel blockers as antihypertensive agents has major health implications. Here, we show that amlodipine has marked intrinsic fluorescence, which further increases considerably inside cells over an identical excitation spectrum as fura-2, confounding the ability to measure cytosolic Ca2+. Using longer wavelength Ca2+ indicators, we find that concentrations of Ca2+ channel blockers that match therapeutic levels in serum of patients do not activate store-operated Ca2+ entry. Antihypertensive Ca2+ channel blockers at pharmacological concentrations either have no effect on store-operated channels, activate them indirectly through store depletion or inhibit the channels. Importantly, a meta-analysis of published clinical trials and a prospective real-world analysis of patients prescribed single antihypertensive agents for 6 mo and followed up 1 yr later both show that dihydropyridines are not associated with increased heart failure or other cardiovascular disorders. Removal of dihydropyridines for treatment of hypertension cannot therefore be recommended.